The ability to site-specifically, chemically modify proteins with nonpeptidic molecules such as spectroscopic probes, catalytic auxilaries, or polymers, or covalently cross-link a protein to another protein or to any other moiety, provides a powerful means to both investigate and manipulate the chemical and biological properties of proteins. A common approach involves the bioconjugation of nucleophilic surface residues on the protein, e.g., the side chains of lysine, histidine, or cysteine, with electrophilic groups on an exogenous molecule, such as aldehydes, α-halo carboxamides, and N-hydroxy succinimides (Lemineux, G. A.; Bertozzi, C. R. TIBTECH 1996, 16, 506).
Unfortunately, a challenge in using the naturally occurring nucleophilic targets in a protein to target modifications is the modest selectivity of these reactions and the multiple occurrences of nucleophilic amino acids in proteins, leading to the formation of heterogeneous mixtures of labeled proteins. Furthermore, the nucleophile-targeted modification reactions frequently require non-physiological conditions, which can preclude in vivo modification strategies and/or result in a loss of protein biological activity.
There is a need in the art to create new targets and novel strategies for specific and targeted protein modifications. Unfortunately, every known organism, from bacteria to humans, encodes the same twenty common amino acids (with the rare exceptions of selenocysteine (see, e.g., A. Bock et al., (1991), Molecular Microbiology 5:515-20) and pyrrolysine (see, e.g., G. Srinivasan, et al., (2002), Science 296:1459-62). This feature limits the use of naturally occurring amino acids in the development of novel chemistries for targeted protein modification.
One strategy to overcome this limitation is to expand the genetic code and add amino acids that have distinguishing chemical properties to the biological repertoire. This approach has proven feasible by the use of “orthogonal” tRNA's and corresponding novel “orthogonal” aminoacyl-tRNA synthetases to add unnatural amino acids to proteins using the in vivo protein biosynthetic machinery of the eubacteria Escherichia coli (E. coli) and other organisms (e.g., Wang et al., (2001), Science 292:498-500; Chin et al., (2002) Journal of the American Chemical Society 124:9026-9027; Chin and Schultz, (2002), ChemBioChem 11:1135-1137; Chin, et al., (2002), PNAS United States of America 99:11020-11024; and Wang and Schultz, (2002), Chem. Comm., 1-10). See also, International Publications WO 2002/086075, entitled “METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; and WO 2005/007624, filed Jul. 7, 2004.
There is a need in the art for novel methods to accomplish highly specific and targeted protein modifications. There is a need in the art for the development of orthogonal translation components that incorporate unnatural amino acids in vivo into proteins in E. coli, where the unnatural amino acids can be incorporated in a defined position, and where the unnatural amino acid has distinguishing chemical properties that allow it serve as a target for specific modification to the exclusion of cross reactions or side reactions with other parts of the proteins. This need in the art is especially applicable to E. coli, as eubacterial protein expression systems can produce large quantities of recombinant protein material for scientific study or therapeutic applications. This invention fulfills these and other needs, as will be apparent upon review of the following disclosure.